1. Field of the Invention
This invention relates to enhancers of the translation of mRNA.
2. Discussion of the Background
The mechanisms by which eukaryotes and prokaryotes initiate translation are known to have certain features in common and to differ in others. Eukaryotic messages are functionally monocistronic, translation initiates at the 5' end and is stimulated by the presence of a cap structure (m.sup.7 G.sup.5' ppp.sup.5' G . . .) at this end (Shatkin, Cell 9 645 (1976)). Prokaryotic messages can be polycistronic, can initiate at sites other than the 5' terminus, and the presence of a cap does not lead to translational stimulation. Both eukaryotes and prokaryotes begin translation at the codon AUG, although prokaryotes can also use GUG. Translation in both is stimulated by certain sequences near the start codon. For prokaryotes, it is the so-called Shine-Dalgarno sequence (a purine rich region 3-10 nucleotides upstream from the initiation codon). For eukaryotes, it is a purine at the -3 position and a G residue in the +4 position (where the A of the AUG start codon is designated +1), plus other sequence requirements involved in finer tuning. This is part of the "relaxed" version of the scanning model (Kozak, Nuc. Acids. Res. 13, 857 (1984)) whereby a 40S ribosomal sub-unit binds at the 5' end of the eukaryotic mRNA and proceeds to scan the sequence until the first AUG, which meets the requirements of the model, is encountered, at which point a 60S sub-unit joins the 40S sub-unit, eventually resulting in protein synthesis. Reference can be made in this connection to the following publications by Kozak: Cell 15, 1109 (1978), Nuc. Acid. Res. 9, 5233 (1981) and Cell 44, 283 (1986).
Beyond these sequence requirements in and about the initiation codon, there are no additional regions of mRNA which are known to enhance translation reproducibly.
During eukaryotic transcription, sequences known as "enhancer" regions can provide transcriptional stimulation.
RNA viruses contain either positive or negative single-stranded RNA or double-stranded RNA. The class of RNA viruses include the majority of plant viruses (over 90%), some animal viruses and several bacteriophage.
One of the most widely studied of RNA viruses is tobacco mosaic virus (referred to hereinafter as TMV). The complete nucleotide sequence of TMV is known (Goelet et al., Proc. Natl. Acad. Sci. USA 79 5818 (1982)). The 5'-region of TMV RNA was first isolated in 1965 as an RNase T.sub.1 -resistant fragment 70 nucleotides long (Mundry, Z. Vererbungsl. 97, 281 (1965)) and free of internal G residues (Mandeles, J. Biol. Chem. 243, 3671). This region (later referred to as omega, Mandeles J. Mol. Biol. Chem. 243 3671) was sequenced (Richards et al. Eur. J. Biochem. 84 513 (1978)) and shown to form disome initiation complexes with wheat germ ribosomes. One ribosome occupies the AUG initiation side of the 126 KDa protein coding region and the second binds to the upstream leader sequence (Filipowicz and Haenni, Proc. Natl. Acad. Sci. USA 76, 3111 (1979); Konarska et al., Eur. J. Biochem. 114, 221 (1981)). Recent additional evidence to support the role of omega in ribosome association comes from the uncoating/gene expression data of Wilson and co-workers, (reviewed in J. Gen. Virol. 66, 1201 (1985) and UCLA Symp. 54 159 (1987)). It was observed in various in vitro translation systems that TMV particles are uncoated by the binding of ribosomes to the 5' end and their translational movement toward the 3' terminus.